Insulin resistance is central to the pathogenesis of Type 2 diabetes (T2DM), the Metabolic Syndrome, and cardiovascular disease, and presents a heavy burden of patient suffering and health care costs. The immediate cause involves defective stimulation of glucose uptake into skeletal muscle. Insulin resistance is also associated with abnormalities in fat oxidation and accumulation of intramyocellular lipid (IMCL), although molecular mechanisms are unknown. Since mitochondria are responsible for lipid oxidation, it is tempting to explain increased IMCL on the basis of mitochondrial dysfunction, and there is recent evidence to support this idea. Over the past grant cycle, we have examined differential gene expression in human muscle using cDNA microarrays and found that multiple genes encoding mitochondrial proteins are down-regulated in insulin resistance. This data, together with the observation that insulin resistance involves selective depletion of complex IV proteins, have led us to a more detailed examination of the mitochondrial proteome and function in skeletal muscle. The current proposal combines human physiology and basic methods to test the hypotheses that: 1) specific depletion of respiratory complex proteins per mitochondrion and a decrease in mitochondrial mass impair substrate oxidation and promote IMCL accumulation in insulin resistance and T2DM;2) these abnormalities increase ROS production which further compromises function in association with oxidative protein modifications, particularly in uncontrolled T2DM. To test these hypotheses, we will study metabolically-characterized insulin sensitive and resistant normoglycemic subjects over a range of body weight, and T2DM patients both in the untreated state and after euglycemic therapy to reverse the "glucose toxicity" component of insulin resistance. Perturbation studies will be performed before and after an insulin-sensitizing thiazolidinedione, and hypocaloric feeding &weight loss, in order to probe relationships with muscle mitochrondrial mass and morphology, and to study respiratory chain proteomics using 2D blue native gel electrophoresis and mass spectrometry. To assess mitochondrial function, we will measure carbohydrate and lipid substrate oxidation by high resolution respirotometry, activity of individual respiratory chain complexes I-IV, coupling status, ROS/RNS generation, and post-translational oxidative modifications affecting mitochondrial proteins. We will also examine whether discordant regulation between nuclear and mitochondrial genomes could underlie mitochondrial dysfunction. These studies will for the first time delineate functional and molecular defects in muscle mitochondria related to defects in substrate oxidation and IMCL in human insulin resistance.